Limits...
Inducible expression of catalytically active type 1 serine/threonine protein phosphatase in a human carcinoma cell line.

Reeder JE, Sowden MP, Messing EM, Klover P, Villa-Moruzzi E, Ludlow JW - Cancer Cell Int. (2003)

Bottom Line: Of critical importance is the question of the in vivo effect of PP1 activity on pRB and growth regulation.When forcibly overexpressing 6His-HA-PP1alpha, there is a concomitant decrease in endogenous PP1alpha levels.Implications of these findings for the study of PP1alpha function in vivo are discussed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York, 14642, USA. jludlow@vestatherapeutics.com

ABSTRACT
BACKGROUND: One of the major cellular serine/threonine protein phosphatases is protein phosphatase type 1 (PP1). Studies employing many eukaryotic systems all point to a crucial role for PP1 activity in controlling cell cycle progression. One physiological substrate for PP1 appears to be the product of the retinoblastoma susceptibility gene (pRB), a demonstrated tumor suppressor. The growth suppressive activity of pRB is regulated by its phosphorylation state. Of critical importance is the question of the in vivo effect of PP1 activity on pRB and growth regulation. As a first step towards addressing this question, we developed an inducible PP1 expression system to investigate the regulation of PP1 activity. RESULTS: We have established a cell line for inducing protein expression of the type 1, alpha-isotype, serine/threonine protein phosphatase (PP1alpha). A plasmid encoding a fusion protein of the catalytic subunit of PP1alpha with a 6-histidine peptide (6His) and a peptide from hemagluttinin (HA) was transfected into the UMUC3 transitional cell carcinoma cell line, previously transfected with the reverse tetracycline transactivator plasmid pUHD172-1neo. A stable cell line designated LLWO2F was established by selection with hygromycin B. 6His-HA-PP1alpha protein appeared in cell lysates within two hours following addition of doxycycline to the culture medium. This protein localizes to the nucleus as does endogenous PP1alpha, and was shown to associate with PNUTS, a PP1-nuclear targeting subunit. Like endogenous PP1alpha, immunocomplexed 6His-HA-PP1alpha is active toward phosphorylase a and the product of the retinoblastoma susceptibility gene, pRB. When forcibly overexpressing 6His-HA-PP1alpha, there is a concomitant decrease in endogenous PP1alpha levels. CONCLUSIONS: These data suggest the existence of an autoregulatory mechanism by which PP1alpha protein levels and activity remain relatively constant. RT-PCR analyses of isolated polysome fractions support the notion that this putative autoregulatory mechanism is exerted, at least in part, at the translational level. Implications of these findings for the study of PP1alpha function in vivo are discussed.

No MeSH data available.


Related in: MedlinePlus

A. Doxycycline has no effect on PP1α protein expression in control cell lines. Equal quantities (50 ug) of whole-cell lysates harvested in the absence (indicated above the lanes with a (-) symbol), or presence (at 24 hr following addition, indicated above the lanes with a (+) symbol), of doxycycline were separated by SDS-PAGE and subjected to immunoblotting using antibody specific for PP1α. UMUC3 – parent cell line from which stable expressor LLWO2F was selected; LLWO1 – cell line generated from UMUC3 which was stably transfected with the reverse tetracycline transactivator plasmid pUHD172-1neo only. B – Doxycycline induces a protein in LLW02F cells that is recognized by antibody to hemagglutinin. Equal quantities (50 ug) of whole-cell lysates harvested at the various time points in hours after doxycycline addition (indicated above each lane) were separated by SDS-PAGE and subjected to immunoblotting as described in Materials and Methods. C – Time-course of recovery from doxycycline induction. LLWO2F cells were induced for 24 hr. At the end of this time, equal quantities (50 ug) of whole-cell lysates harvested at the various time points in hours following removal of doxycycline (indicated above each lane) were separated by SDS-PAGE and subjected to immunoblotting with antibody to hemagglutinin.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC183861&req=5

Figure 2: A. Doxycycline has no effect on PP1α protein expression in control cell lines. Equal quantities (50 ug) of whole-cell lysates harvested in the absence (indicated above the lanes with a (-) symbol), or presence (at 24 hr following addition, indicated above the lanes with a (+) symbol), of doxycycline were separated by SDS-PAGE and subjected to immunoblotting using antibody specific for PP1α. UMUC3 – parent cell line from which stable expressor LLWO2F was selected; LLWO1 – cell line generated from UMUC3 which was stably transfected with the reverse tetracycline transactivator plasmid pUHD172-1neo only. B – Doxycycline induces a protein in LLW02F cells that is recognized by antibody to hemagglutinin. Equal quantities (50 ug) of whole-cell lysates harvested at the various time points in hours after doxycycline addition (indicated above each lane) were separated by SDS-PAGE and subjected to immunoblotting as described in Materials and Methods. C – Time-course of recovery from doxycycline induction. LLWO2F cells were induced for 24 hr. At the end of this time, equal quantities (50 ug) of whole-cell lysates harvested at the various time points in hours following removal of doxycycline (indicated above each lane) were separated by SDS-PAGE and subjected to immunoblotting with antibody to hemagglutinin.

Mentions: Figure 1 shows construct of the tetracycline-inducible plasmid used to express 6His-HA-PP1α. As shown in Figure 2A, 24 hr incubation with doxycycline in the medium has no appreciable effect on endogenous PP1α protein expression in the untransfected parent cell line (UMUC3) or a cell line transfected with the reverse tetracycline transactivator plasmid pUHD172-1neo only (LLWO1). A time course of induction by doxycycline was then carried out in stable clone LLWO2F. Harvested at various time intervals, whole-cell lysates were prepared and the proteins were separated by SDS-PAGE for western blotting using antibody to the hemagglutinin tag. As shown in Figure 2B, a protein with the anticipated molecular weight of 40 kDa was detected. A clear differential of expression of 6His-HA-PP1α (indicated at the left of the panel) was observed at 1.5 hours after addition of doxycycline, which became more pronounced at later time points. The doxycycline-independent increase in 6His-HA-PP1α is attributed to background expression when using this system [25]. As predicted for a protein under the control of the doxycycline-inducible promoter, the abundance of 6His-HA-PP1α decreases in the absence of the inducer over time (Figure 2C). Subsequent time course experiments revealed peak abundance occurring between 16 and 24 hr, and remaining constant thereafter for at least 72 hr, at which time the experiment was terminated. As such, we have chosen the 24 hr induction time for all subsequent experiments to ensure peak abundance of the induced 6His-HA-PP1α.


Inducible expression of catalytically active type 1 serine/threonine protein phosphatase in a human carcinoma cell line.

Reeder JE, Sowden MP, Messing EM, Klover P, Villa-Moruzzi E, Ludlow JW - Cancer Cell Int. (2003)

A. Doxycycline has no effect on PP1α protein expression in control cell lines. Equal quantities (50 ug) of whole-cell lysates harvested in the absence (indicated above the lanes with a (-) symbol), or presence (at 24 hr following addition, indicated above the lanes with a (+) symbol), of doxycycline were separated by SDS-PAGE and subjected to immunoblotting using antibody specific for PP1α. UMUC3 – parent cell line from which stable expressor LLWO2F was selected; LLWO1 – cell line generated from UMUC3 which was stably transfected with the reverse tetracycline transactivator plasmid pUHD172-1neo only. B – Doxycycline induces a protein in LLW02F cells that is recognized by antibody to hemagglutinin. Equal quantities (50 ug) of whole-cell lysates harvested at the various time points in hours after doxycycline addition (indicated above each lane) were separated by SDS-PAGE and subjected to immunoblotting as described in Materials and Methods. C – Time-course of recovery from doxycycline induction. LLWO2F cells were induced for 24 hr. At the end of this time, equal quantities (50 ug) of whole-cell lysates harvested at the various time points in hours following removal of doxycycline (indicated above each lane) were separated by SDS-PAGE and subjected to immunoblotting with antibody to hemagglutinin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC183861&req=5

Figure 2: A. Doxycycline has no effect on PP1α protein expression in control cell lines. Equal quantities (50 ug) of whole-cell lysates harvested in the absence (indicated above the lanes with a (-) symbol), or presence (at 24 hr following addition, indicated above the lanes with a (+) symbol), of doxycycline were separated by SDS-PAGE and subjected to immunoblotting using antibody specific for PP1α. UMUC3 – parent cell line from which stable expressor LLWO2F was selected; LLWO1 – cell line generated from UMUC3 which was stably transfected with the reverse tetracycline transactivator plasmid pUHD172-1neo only. B – Doxycycline induces a protein in LLW02F cells that is recognized by antibody to hemagglutinin. Equal quantities (50 ug) of whole-cell lysates harvested at the various time points in hours after doxycycline addition (indicated above each lane) were separated by SDS-PAGE and subjected to immunoblotting as described in Materials and Methods. C – Time-course of recovery from doxycycline induction. LLWO2F cells were induced for 24 hr. At the end of this time, equal quantities (50 ug) of whole-cell lysates harvested at the various time points in hours following removal of doxycycline (indicated above each lane) were separated by SDS-PAGE and subjected to immunoblotting with antibody to hemagglutinin.
Mentions: Figure 1 shows construct of the tetracycline-inducible plasmid used to express 6His-HA-PP1α. As shown in Figure 2A, 24 hr incubation with doxycycline in the medium has no appreciable effect on endogenous PP1α protein expression in the untransfected parent cell line (UMUC3) or a cell line transfected with the reverse tetracycline transactivator plasmid pUHD172-1neo only (LLWO1). A time course of induction by doxycycline was then carried out in stable clone LLWO2F. Harvested at various time intervals, whole-cell lysates were prepared and the proteins were separated by SDS-PAGE for western blotting using antibody to the hemagglutinin tag. As shown in Figure 2B, a protein with the anticipated molecular weight of 40 kDa was detected. A clear differential of expression of 6His-HA-PP1α (indicated at the left of the panel) was observed at 1.5 hours after addition of doxycycline, which became more pronounced at later time points. The doxycycline-independent increase in 6His-HA-PP1α is attributed to background expression when using this system [25]. As predicted for a protein under the control of the doxycycline-inducible promoter, the abundance of 6His-HA-PP1α decreases in the absence of the inducer over time (Figure 2C). Subsequent time course experiments revealed peak abundance occurring between 16 and 24 hr, and remaining constant thereafter for at least 72 hr, at which time the experiment was terminated. As such, we have chosen the 24 hr induction time for all subsequent experiments to ensure peak abundance of the induced 6His-HA-PP1α.

Bottom Line: Of critical importance is the question of the in vivo effect of PP1 activity on pRB and growth regulation.When forcibly overexpressing 6His-HA-PP1alpha, there is a concomitant decrease in endogenous PP1alpha levels.Implications of these findings for the study of PP1alpha function in vivo are discussed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York, 14642, USA. jludlow@vestatherapeutics.com

ABSTRACT
BACKGROUND: One of the major cellular serine/threonine protein phosphatases is protein phosphatase type 1 (PP1). Studies employing many eukaryotic systems all point to a crucial role for PP1 activity in controlling cell cycle progression. One physiological substrate for PP1 appears to be the product of the retinoblastoma susceptibility gene (pRB), a demonstrated tumor suppressor. The growth suppressive activity of pRB is regulated by its phosphorylation state. Of critical importance is the question of the in vivo effect of PP1 activity on pRB and growth regulation. As a first step towards addressing this question, we developed an inducible PP1 expression system to investigate the regulation of PP1 activity. RESULTS: We have established a cell line for inducing protein expression of the type 1, alpha-isotype, serine/threonine protein phosphatase (PP1alpha). A plasmid encoding a fusion protein of the catalytic subunit of PP1alpha with a 6-histidine peptide (6His) and a peptide from hemagluttinin (HA) was transfected into the UMUC3 transitional cell carcinoma cell line, previously transfected with the reverse tetracycline transactivator plasmid pUHD172-1neo. A stable cell line designated LLWO2F was established by selection with hygromycin B. 6His-HA-PP1alpha protein appeared in cell lysates within two hours following addition of doxycycline to the culture medium. This protein localizes to the nucleus as does endogenous PP1alpha, and was shown to associate with PNUTS, a PP1-nuclear targeting subunit. Like endogenous PP1alpha, immunocomplexed 6His-HA-PP1alpha is active toward phosphorylase a and the product of the retinoblastoma susceptibility gene, pRB. When forcibly overexpressing 6His-HA-PP1alpha, there is a concomitant decrease in endogenous PP1alpha levels. CONCLUSIONS: These data suggest the existence of an autoregulatory mechanism by which PP1alpha protein levels and activity remain relatively constant. RT-PCR analyses of isolated polysome fractions support the notion that this putative autoregulatory mechanism is exerted, at least in part, at the translational level. Implications of these findings for the study of PP1alpha function in vivo are discussed.

No MeSH data available.


Related in: MedlinePlus